The DNA dependent protein kinase catalytic subunit (DNA-PKcs) and its DNA-binding partner the Ku70/80 heterodimer are the key components of the non-homologous end-joining (NHEJ) pathway. In response to DNA double-strand breaks (DSBs), DNA-PKcs is rapidly phosphorylated at the T2609 cluster, an event critical for DSB repair. DNA-PKcs 3A knockin mice, in which phosphorylation at three residues in the mouse T2605 cluster (human T2609 cluster) are ablated via alanine substitution, die prematurely due to congenital bone marrow failure. Loss of hematopoietic stem cells (HSCs) in DNA-PKcs3A/3A mice is caused by elevated genotoxic stress as evidenced by increased intestinal crypt apoptosis and skin hyperpigmentation. Although these mice die prematurely, they can be rescued with bone marrow transplantation. BMT-rescued DNA- PKcs3A/3A mice are prone to develop both hematologic and non-hematologic cancers. HSC loss and hyperpigmented skin are the main features found in human dyskeratosis congenita (DC) syndrome and in mice double knockout of protection of telomeres 1b (POT1b) and telomerase RNA (mTR) genes. In addition, DC patients are also prone to cancer development. DC patients and POT1b/mTR DKO mice are unable to properly maintain the telomeres. Based on these findings, we hypothesize that the expression of the DNA-PKcs3A protein will lead to telomere dysregulation, genome instability, and carcinogenesis. We propose in this project to further elucidate the mechanism by which DNA-PKcs T2609 cluster phosphorylation impacts HSC homeostasis and telomere maintenance. Our specific aims are: 1. To investigate how DNA-PKcs T2609 cluster phosphorylation and the DNA-PKcs interaction with the Ku70/80 heterodimer impacts hematopoietic stem cell homeostasis. 2. To investigate the effect of DNA-PKcs T2609 cluster phosphorylation on telomere maintenance. 3. To determine the effect of DNA-PKcs T2609 cluster phosphorylation on the production and protection of telomeric 3' overhangs.